In recent years, as a DNA amplification method of amplifying a particular DNA fragment rapidly and easily, a polymerase chain reaction (PCR) method is used in every organism-related field. The PCR method is a method of designing two primers complementary with a template DNA, and replicating a region held by the primers in vitro. The method is to obtain a PCR product by amplifying a DNA exponentially by repeating a temperature cycle of incubating a reaction solution containing a template DNA, a primer, a nucleotide, and a heat-resistant DNA polymerase at various temperatures.
One time cycle including (1) denaturing a double-stranded DNA into single-stranded DNAs, (2) annealing primers to the single-stranded DNA, and (3) incubating it at respective temperature conditions (94° C., 50 to 60° C., and 74° C., respectively) under which a DNA chain complementary with the single-stranded chain is synthesized to extend a DNA, for a container containing a template DNA, a primer, a DNA polymerase, a nucleotide and a reaction buffer, thereby, and one molecule of a DNA fragment is rendered into two molecules. In the next cycle, since the DNA fragment synthesized in the previous cycle becomes a template, DNA fragments synthesized after n cycles become 2n molecules.
Previously, in controlling of a temperature, a container made of a glass containing a template DNA, a primer, a DNA polymerase, a nucleotide and a reaction buffer is accommodated in an accommodating part of a block-like constant temperature device formed of a material of aluminum or the like, the block-like accommodation part made of a metal is heated or cooled, and one waits until a liquid temperature becomes an equal temperature distribution, thereby, next heating or cooling is performed (Patent Document 1).
For this reason, there is a problem that until a reaction solution in the container is heated or cooled, it takes a time to obtain equal liquid temperature distribution due to great volume of the container and, at the same time, complicated temperature change is generated due to a difference in heat capacity and specific heat between the accommodation part and the container, and it is necessary to perform complicated temperature controlling in order to amplify a DNA at a high precision.
Meanwhile, in the PCR method, controlling of a temperature is important, and quality and an amount of the finally obtained PCR product can be changed by changing a temperature cycle.
Particularly, in real time PCR, more precise quantitation is performed by detecting a process of producing an amplification product by PCR at real time, and analyzing this, and it is necessary to control a temperature at a higher precision and rapidly. For this reason, various apparatuses are proposed (Patent Document 2 to Patent Document 5). However, these apparatuses are large scale complicated apparatuses in which a complicated flow path is provided, and a large-scale centrifugation apparatus is used.
To the contrary, the present inventor disclosed a reaction container including a reaction container body provided with a reaction chamber accommodating a reaction solution, and a lid material capable of sealing an opening of the reaction chamber, wherein the lid material has a pressing part for pressing the reaction solution, thereby, it enabled to rapidly control a temperature at a scale of a simple apparatus without necessity of a centrifugal force (Patent Document 6).
In addition, the present inventor enabled to simultaneously shorten and automate a series of treatments with respect to PCR or the like without using a large-scale apparatus, by thinning or capillarizing a liquid having a high heat efficiency, and connecting reasonable centrifugation treatment and suction discharge treatment based on an especial shape of the container (Patent Document 7).
On the other hand, as an apparatus for implementing the PCR method, previously, an apparatus of applying a centrifugal force to introduce a liquid into a container in which a tube-like tip capillarized for enhancing following capability of thermal response is closed and control a temperature has been known. However, in the previous apparatus, since a bottom of the container is closed, it is difficult to completely remove the air, therefore, the apparatus has a problem that the air remaining on a bottom of the container may be dilated to fly the liquid introduced into the container to the inside and the outside of the container. In addition, the apparatus also has a problem that the gas contained in the liquid itself causes foaming, and this may impede uniform temperature controlling.
Then, the present inventor got an idea that, by utilizing the previously used dispensing apparatus having a nozzle with an open tip which enables suction and discharge of quantitation of an amount of a liquid, shortening of a treating time of, and automation of temperature controlling treatment for PCR, a series of treatments including light measurement, can be simultaneously performed at high reliance, without using a special container resulting from thinning or capillarizing a liquid, and without necessity of centrifugation treatment.
[Patent Document 1] Japanese Patent No. 2622327
[Patent Document 2] Japanese Patent Application National Publication (Laid-Open) No. 2000-511435
[Patent Document 3] Japanese Patent Application National Publication (Laid-Open) No. 2003-500674
[Patent Document 4] Japanese Patent Application National Publication (Laid-Open) No. 2003-502656
[Patent Document 5] U.S. Pat. No. 5,958,349
[Patent Document 6] Japanese Patent Application Laid-Open No. 2002-10777
[Patent Document 7] International Publication WO 2006/038643